Background There is increasing evidence to support a role for the gastrointestinal microbiota in the etiology of irritable bowel syndrome (IBS). Given the evidence of an inflammatory component to IBS, the mucosa-associated microbiota potentially play a key role in its pathogenesis. The objectives were to compare the mucosa-associated microbiota between patients with diarrhea predominant IBS (IBS-D), constipation predominant IBS (IBS-C) and controls using fluorescent in situ hybridization and to correlate specific bacteria groups with individual IBS symptoms. Methods Forty-seven patients with IBS (27 IBS-D and 20 IBS-C) and 26 healthy controls were recruited to the study. Snap-frozen rectal biopsies were taken at colonoscopy and bacterial quantification performed by hybridizing frozen sections with bacterial-group specific oligonucleotide probes. Key Results Patients with IBS had significantly greater numbers of total mucosa-associated bacteria per mm of rectal epithelium than controls [median 218 (IQR - 209) vs 128 (121) P = 0.007], and this was chiefly comprised of bacteroides IBS [69 (67) vs 14 (41) P = 0.001] and Eubacterium rectale-Clostridium coccoides [52 (58) vs 25 (35) P = 0.03]. Analysis of IBS sub-groups demonstrated that bifidobacteria were lower in the IBS-D group than in the IBS-C group and controls [24 (32) vs 54 (88) vs 32 (35) P = 0.011]. Finally, amongst patients with IBS, the maximum number of stools per day negatively correlated with the number of mucosa-associated bifidobacteria (P < 0.001) and lactobacilli (P = 0.002). Conclusions & Inferences The mucosa-associated microbiota in patients with IBS is significantly different from healthy controls with increases in bacteroides and clostridia and a reduction in bifidobacteria in patients with IBS-D.